Project Summary Potential roles of neutrophils and platelets in the pathophysiology of intracranial aneurysms have been long suggested by clinical observations. However, the exact mechanism by which neutrophils and platelets work to promote the rupture of intracranial aneurysms is not well understood. Activated neutrophils can release decondensed DNA decorated with neutrophil enzymes such as neutrophil elastase and myeloperoxidase, resulting in the formation of Neutrophil Extracellular Traps (NETs) and the death of neutrophils (NETosis). NETs were originally described as a structure that can trap and dissolve pathogens. However, recent studies suggest roles of NETs in sterile diseases that involve vascular inflammation including atherosclerosis, vasculitis, and venous thrombosis. Interactions between NETs and platelets may result in a vicious cycle of NET formation and platelet activation that leads to the excessive vascular wall damages and aneurysmal rupture. Our preliminary studies show that NETs exist in both human intracranial aneurysms and experimental mouse aneurysms. In addition, mice lacking protein arginine deiminases-4 (PAD4), an enzyme required for the NET formation, had a reduced rupture rate. We hypothesize that NETs, neutrophil extracellular traps, can promote the development of intracranial aneurysm rupture through the vicious cycle of neutrophil and platelet activation. In Aim 1, we will test neutrophil PAD4-deficient mice have a lower rupture rate and reduce NET formations compared to the control mice. In Aim 2, we will test whether the pharmacological prevention of NET formation by PAD4 inhibitors (Cl-amidine and GSK484) or the resolution of NETs by deoxyribonuclease reduces aneurysmal rupture. In Aim 3, we will test whether platelets and neutrophil-platelet interactions contribute to aneurysmal rupture. We will test whether thrombocytopenic mice have reduced NET formations and lower rupture rates. We will also study the contribution of P-selectin or CD11b/CD18-mediated neutrophil-platelet interactions to the development of aneurysmal rupture. The proposed studies will provide new insights into the roles of NETs and their interactions with platelets in the development of aneurysmal rupture. The results will be a basis for future studies to develop new therapies that target NETs and platelet-neutrophil interactions for the prevention of aneurysmal rupture.